Major Cryopreservation Variables

Freezing-compatible pH buffers

Vehicle solution selection (may vary with cryoprotectant selection)

Apoptosis inhibitors (may be required to get long-term post-thaw cell survival for some cells)

Cryoprotectant selection (optima may vary with vehicle solution selected)

Cooling rate

Storage temperature

Warming rate

Cryoprotectant addition/elution conditions (number of steps, temperature)

optimum preservation at reduced temperatures and we have long advocated the use of intracellular-type solutions as more appropriate vehicle solutions for CPAs.77 81 116-118 Maintaining the ionic and hydraulic balance within tissues during cold exposure can be better controlled in media designed to physically restrict these temperature-induced imbalances and can be applied equally to the choice of vehicle solution for adding and removing CPAs in a cryopreservation protocol.119 Moreover, the nature of the vehicle solution used to expose cells and tissues to cryoprotectants at low temperatures has been shown to impact the outcome of cryopreservation,77 117 120 121 and has recently become the focus of additional research aimed at optimization and attenuation of the so-called cryopreservation cap 80,119,122-124

Figure 8.2 illustrates the marked effect that an intracellular-type vehicle solution can have on the outcome of cryopreservation. In a study of factors that influence the survival of vascular smooth muscle and endothelial cells it was discovered that the choice of carrier solution significantly impacted the optimum survival of the cells. Moreover, the survival varied with the nature of the CPA and the cell type suggesting that nature of the vehicle solution should be included as one of the variables that must be optimized for a given system. Our aim is to use the approach of a hybrid universal formulation in an attempt to nullify the wide differences in available solution choices. Baust et al. have corroborated this approach and shown that an intracellular-type solution, Hypothermosol, provides a significantly better vehicle solution for CPAs than a range of extracellular-type media in other cell systems.123-125

Another common practice in tissue banking is to employ serum of animal origin in the cryopreservation formulation. Serum-free procedures have been reported for a variety of tissues126 and mammalian sources of serum can be removed providing that cryopreservation conditions are subsequently reoptimized.127

8.4.1 Synthetic Cryoprotectants

Historically, serendipity has been largely responsible for most discoveries of cryoprotectants. Cryoprotectant selection for cryopreservation in general is usually restricted to those that have conferred cryoprotection in a variety of biological systems (dextrans, DMSO, ethylene glycol, glycerol, hydroxyethyl starch, polyvinylpyrrolidone, sucrose, and trehalose).10 Combinations of two cryoprotectants may result in additive or synergistic enhancement of cell survival.128 129 Comparison of chemicals with cryoprotectant properties has revealed no common structural features. These chemicals are usually divided into two classes: (1) intracellular cryoprotectants with low molecular weights that penetrate and permeate cells and (2) extracellular cryoprotectants with relatively high molecular weights (greater than or equal to sucrose [342 daltons]) that neither penetrate nor permeate cells. A variety of biologic chemicals with cryoprotective activity for one or more biological systems have been reported (Table 8.3).

Intracellular cryoprotectants, such as glycerol and DMSO at concentrations from 0.5 to 3.0 molar, are effective in minimizing cell damage in slowly frozen biological systems. Extracellular

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